Seasonal variation in chemical composition of size-segregated urban air particles and the inflammatory activity in the mouse lung.

We investigated the seasonal variations in the chemical composition and in vivo inflammatory activity of urban air particulate samples in four size ranges (PM(10-2.5), PM(2.5-1), PM(1-0.2), and PM(0.2)). The samples were collected in Helsinki using a high-volume cascade impactor (HVCI). Healthy C57BL/6J mice were intratracheally instilled with a single dose (10 mg/kg) of the particulate samples. The lungs were lavaged and the bronchoalveolar lavage fluid (BALF) was assayed for indicators of inflammation and tissue damage: cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6, and keratinocyte-derived chemokine [KC]) at 4 h, and total cell number and total protein concentration at 12 h. The PM(10-2.5) and PM(2.5-1) samples had much higher inflammatory potency than the PM(1-0.2) and PM(0.2) samples. The relative inflammatory activities of the autumn samples were the highest on an equal mass basis, but when estimated for the particulate mass per cubic meter of air, the springtime samples had the highest inflammatory potential. Resuspended soil material and other non-exhaust particulate material from traffic were associated with a high inflammatory activity of the PM(10-2.5) and PM(2.5-1) samples. Secondary inorganic ions in the PM(1-0.2) and PM(0.2) samples had inconsistent negative or positive correlations with the inflammatory activity. There were no systematic seasonal variations in the tracers of incomplete combustion and atmospherically oxidized organics in the PM(1-0.2) and PM(0.2) samples, which probably explains their low correlations with the inflammatory activity. In conclusion, in a relatively clean Nordic city, the resuspension of road dust and other non-exhaust particulate material from traffic were the major sources of inflammatory activity of urban air inhalable particles.

Inflammation and tissue damage in mouse lung by single and repeated dosing of urban air coarse and fine particles collected from six European cities.

The authors have previously demonstrated heterogeneities in the inflammatory activities of urban air fine (PM(2.5-0.2)) and coarse (PM(10-2.5)) particulate samples collected from six European cities with contrasting air pollution situations. The same samples (10 mg/kg) were intratracheally instilled to healthy C57BL/6J mice either once or repeatedly on days 1, 3, and 6 of the study week. The lungs were lavaged 24 h after the single dose or after the last repeated dosing. In both size ranges, repeated dosing of particles increased the total cell number in bronchoalveolar lavage fluid (BALF) more than the respective single dose, whereas cytokine concentrations were lower after repeated dosing. The lactate dehydrogenase (LDH) responses increased up to 2-fold after repeated dosing of PM(2.5-0.2) samples and up to 6-fold after repeated dosing of PM(10-2.5) samples. PM(10-2.5) samples evoked a more extensive interstitial inflammation in the mouse lungs. The constituents with major contributions to the inflammatory responses were oxidized organic compounds and transition metals in PM(2.5-0.2) samples, Cu and soil minerals in PM(10-2.5) samples, and Zn in both size ranges. In contrast, poor biomass and coal combustion were associated with elevated levels of polycyclic aromatic hydrocarbons (PAHs) and a consistent inhibitory effect on the inflammatory activity of PM(2.5-0.2) samples. In conclusion, repeated intratracheal instillation of both fine and coarse particulate samples evoked enhanced pulmonary inflammation and cytotoxicity compared to single-dose administration. The sources and constituents of urban air particles responsible for these effects appear to be similar to those encountered in the authors' previous single-dose study.

Dose and time dependency of inflammatory responses in the mouse lung to urban air coarse, fine, and ultrafine particles from six European cities.

We investigated the dose and time dependency of inflammatory and cytotoxic responses to size-segregated urban air particulate samples in the mouse lung. Coarse (PM10-2.5), fine (PM2.5-0.2), and ultrafine (PM0.2) particles were collected in six European cities (Duisburg, Prague, Amsterdam, Helsinki, Barcelona, Athens) in selected seasons using a modified Harvard high-volume cascade impactor. Healthy C57Bl/6J mice were intratracheally exposed to the particulate samples in a 24-h dose-response study (1, 3, and 10 mg/kg) and in 4-, 12-, and 24-h time course studies (10 mg/kg). After the exposures, the lungs were lavaged and the bronchoalveolar lavage fluid (BALF) was assayed for indicators of inflammation and tissue damage: total cell number, cell differential, total protein, and lactate dehydrogenase (LDH) and cytokine (tumor necrosis alpha [TNF-alpha], interleukin-6 [IL-6], and keratinocyte-derived chemokine [KC]) concentrations. In general, PM10-2.5 samples had higher inflammatory activity than PM2.5-0.2 samples. PM0.2 samples showed negligible inflammatory activity. PM10-2.5 and PM2.5-0.2 samples caused large increases in BALF cytokine concentrations at 4 h, but not at 12 or 24 h, after exposure. The BALF total cell number and total protein concentrations increased significantly at 12 h for both the PM10-2.5 and PM2.5-0.2 samples, but only PM10-2.5 samples produced consistent, significant increases at 24 h after exposure. There was more heterogeneity in BALF cytokine and neutrophil cell number responses to PM2.5-0.2 samples than to PM10-2.5 samples between the sampling campaigns. Thus, particle size, sources, and atmospheric transformation processes affect the inflammatory activity and response duration of urban air particulate matter in the mouse lung.

A Chemical and Toxicological Comparison of Urban Air PM10 Collected During Winter and Spring in Finland.

We have used a new high-volume, low-cutoff inertial impactor (HVLI) in a pilot study on chemical characterization and toxicity testing of ambient air PM10 in Helsinki, Finland. Ambient air PM10 was collected at 1100 L/min in 2- to 4-day periods. Two different PM10 samples were selected to represent wintertime combustion type and springtime resuspension type particulate matter (PM) pollution. The most abundant water-soluble ions and elements were analyzed by ion chromatography and inductively coupled plasma mass spectrometry, respectively. The proinflammatory activation ¡NO and interleukin 6 (IL-6) production] and viability of cultured murine RAW 264.7 macrophages were tested in 24-h incubations with increasing mass doses (30-2000 µg per 10(6) cells) from the collected PM10 samples. The winter sample had a higher assessed PM2.5 fraction and sulfate content, and lower chloride, sodium, calcium, aluminum, copper, manganese, and especially iron contents than the spring sample. Both PMjo samples induced dose-dependent NO production in murine macrophages, and the springtime PM10 produced also a strong, dose-dependent IL-6 production. In conclusion, the HVLI proved to be a suitable technique for short-term collection of relatively large ambient air PM masses, enabling extensive chemical characterization and toxicity testing from the same samples.

Effects of sample preparation on chemistry, cytotoxicity, and inflammatory responses induced by air particulate matter.

Methanol is used for high-efficiency extraction of air particulate (PM) mass from the sampling substrate in the high-volume cascade impactor. Sonication is needed during extraction and when dissolving dried PM samples in liquids used in exposure studies. We investigated the effects of these procedures on the PM chemistry and PM-induced cytotoxic and inflammatory responses in mouse macrophages. Untreated and methanol-treated ambient air reference PM samples (SRM1649a, EHC-93) and diesel PM (SRM1650) were tested after different sonication durations (530 min). Furthermore, the time dependency of the responses to SRM1649a, EHC-93, and a fine PM sample from Helsinki was investigated. Methanol pretreatment increased on average by 24% and 21% the recovery of water-soluble metals from SRM1649a and EHC-93, but not SRM1650. It had no systematic effect on the recoveries of inorganic secondary ions (NO3-, SO4(2-), NH4+) or the sum of genotoxic PAH compounds from the three reference samples. Nitric oxide (NO) response to SRM1650 was strongly enhanced by methanol pretreatment, whereas the cytotoxic or inflammatory responses to the ambient air PM samples (EHC-93, SRM1649a) were only slightly modified. Sonication duration was a modifying factor only in connection to SRM1650. Maximal interleukin (IL)-1 production was observed earlier (8 h) than maximal tumor necrosis factor (TNF) alpha and IL-6 productions (24 h), which indicates the importance to know the optimal time points for measurement of the selected response parameters. In conclusion, methanol extraction and reasonable sonication duration are not likely to modify the cytotoxic and inflammatory potency of ambient air PM samples, but some responses to air PM, rich in organic compounds, can be modified.

Characterization of respiratory exposure to and effects of cold-air hyperventilation in guinea pigs.

The purpose of this study was to characterize the respiratory effects of single and repeated controlled exposures to clean warm humid and cold dry air in a new model of anesthetized, mechanically ventilated guinea pigs, and to compare findings with known effects in humans. Intratracheal air (T(tr)) and retrotracheal tissue (T(oe)) temperatures and peak expiratory airflow (PEF), tidal volume (V(T)), heart rate, and blood pressure of hyperventilating animals were measured continuously. Four consecutive 10-min exposures to warm humid air (n = 7) produced slight airway warming and minimal lung function changes during the exposure. In a single 10-min exposure to cold dry air (n = 39), T(tr) decreased from (means +/- SEM) 36.1 +/- 0.3 degrees C to 26.3 +/- 0.3 degrees C (Delta = -9.8 +/- 0.4 degrees C) and T(oe) from 36.4 +/- 0.2 degrees C to 35.5 +/- 0.2 degrees C (Delta = -0.9 +/- 0.1 degrees C). PEF and V(T) decreased in response to airway cooling with maximal decrements within the first 2-4 min from the beginning of the exposure period. The maximal decrease in PEF was from 21.7 +/- 0.3 ml s(-1) to 15.9 +/- 0.5 ml s(-1) (Delta = -26.7%) and that in V(T) from 5.2 +/- 0.1 ml to 4.2 +/- 0.1 ml (Delta = -19.2%) (p <.05 for both changes). The decreases in lung functions attenuated significantly during the course of the 10-min exposure to cold dry air, indicating adaptation. Consequently, the decrements in PEF and V(T) at 5, 7.5, and 10 min were significantly smaller than those at 3 min. In four consecutive 10-min exposures to cold dry air (n = 15), there were no statistically significant differences in T(tr) or T(oe) decreases between the exposure periods. The largest decreases in the lung function parameters were during the first exposure period, whereas there were significantly smaller responses during the second and third exposure periods (p <.05). Thus, a highly reproducible airway cooling and an immediate bronchoconstriction were produced in response to cold dry air hyperventilation in guinea pigs. During the course of cold-air exposure and in repeated exposures, there was a significant attenuation of the bronchial response, which resembled the refractoriness of the asthmatic airways to repeated hyperventilation of cold or warm dry air. The present guinea pig model seems to be well suited for production of complementary animal data on the pathophysiological effects of cold dry air on the tracheobronchial airways.

Combined respiratory effects of cold air with SO(2) or NO(2) in repeated 10-minute exposures of hyperventilating guinea pigs.

Previous studies in asthmatic subjects and guinea pigs have demonstrated attenuation of bronchoconstriction in repeated exposures to clean cold dry air. In the present animal study, we have simulated short-lasting human exposures to subfreezing urban air containing sulfur dioxide (SO(2)) and nitrogen dioxide (NO(2)). The anesthetized, paralyzed, and mechanically ventilated guinea pigs had 4 consecutive 10-min exposures either to clean cold dry air or to cold air with graded concentrations of SO(2) (0-5 ppm) or NO(2) (0-4 ppm). Peak expiratory flow (PEF) and tidal volume (V(T)) were continuously measured both during and after highly controlled exposures. Bronchoalveolar lavage fluid (BALF) and histological samples were obtained after finishing the consecutive exposures. Cold air + SO(2) at 1 and 2.5 ppm (n = 12) produced immediate concentration-dependent increases in the lung function responses compared to the preceding single exposure to clean cold dry air in the same animals (DeltaPEF = -32.7 +/- 6.1% and -35.6 +/- 6.5% vs. -27.0 +/- 3.1%; DeltaV(T) = -22.4 +/- 4.4% and -28.3 +/- 4.7% vs. -18.1 +/- 2.9%). In a multivariate analysis, these responses were significantly larger than the attenuated lung function responses to the corresponding second and third clean cold dry air exposures (p <. 05). The fourth exposure to cold air + SO(2) at 5 ppm produced a smaller response (DeltaPEF = -25.3 +/- 4.8% and DeltaV(T) = -17.8 +/- 3.7%) than cold air with the lower SO(2) concentrations. Cold air + NO(2) at 1 and 2.5 ppm (n = 12) produced roughly similar lung function responses to the preceding single exposure to clean cold dry air in the same animals, and there was no significant attenuation of bronchoconstriction as with the consecutive exposures to clean cold dry air. The largest decreases in lung functions (DeltaPEF = -33.8 +/- 6.7% and DeltaV(T) = -26.2 +/- 6.8%) were recorded during the fourth exposure, which was to cold air + NO(2) at 4 ppm. In the cold air + SO(2) group, there was a significantly lower proportion of macrophages in the differential count of BALF white cells compared to the clean cold dry air group. In addition, there was eosinophilic infiltration within and below the tracheal epithelium in all guinea pigs exposed to either clean cold dry air, cold air + SO(2), or cold air + NO(2). In conclusion, the addition of moderate concentrations of SO(2) or NO(2) to clean cold dry air counteracted the attenuation of bronchoconstriction induced by repeated cold dry air exposures in guinea pigs. Cold air + SO(2) also decreased the proportion of macrophages in BALF white cells.

Combined respiratory effects of cold air with SO(2) or NO(2) in single 1-hour exposures of hyperventilating guinea pigs.

The present study is a continuation of our previous experiments on repeated 10-min exposures of anesthetized, mechanically ventilated guinea pigs to clean cold dry air (Hälinen et al., 2000a), and to cold air plus gaseous air pollutants (Hälinen et al., 2000b). This time we made continuous 60-min exposures to clean cold dry air, cold air + SO(2) at 1 ppm, cold air + NO(2) at 1 ppm, and warm humid air + NO(2) at 1 ppm, and focused on responses at 10-60 min. Clean cold dry air and cold air + pollutants (n = 8-9 in each group) produced similar cooling in the guinea pig lower respiratory tract. The decreases in intratracheal temperature (T(tr)) reached a plateau at 20 min with mean maximal decreases of 9.7-11.3 degrees C from the pre-exposure control values of 36.0-37.3 degrees C. In contrast, there were progressive decreases in esophageal temperature (T(oe)) during the exposures, indicating constant conductive and evaporative heat losses from the tracheobronchial tissues. The mean maximal decreases in T(oe) were 1.2-1.4 degrees C from the preexposure control values of 37.8-38.0 degrees C. Clean cold dry air induced 4. 5-10.8% mean decreases in peak expiratory flow (PEF) at 10-60 min of exposure, which were statistically nonsignificant due to a relatively large variation between animals. Cold air + SO(2) at 1 ppm induced a mean decrease of 11.4% in PEF at 10 min (p <.05), which was spontaneously abolished during the next 10 min of exposure. Cold air + NO(2) at 1 ppm caused no decrease, but in fact small, nonsignificant increases in PEF at 30-60 min of exposure. Cold air + NO(2) at 1 ppm, and to some extent also cold air + SO(2) at 1 ppm, attenuated significantly the mechanical ventilation induced gradual decrease in tidal volume (V(T)), when compared to clean cold dry air exposure. Cold air + NO(2) at 1 ppm, but not warm humid air + NO(2) at 1 ppm, increased significantly the proportion of macrophages in the differential count of bronchoalveolar lavage fluid (BALF) white cells when compared to both clean warm humid air and cold dry air. None of the exposure conditions caused morphological or inflammatory changes in the respiratory tissues. In conclusion, continuous 60-min exposures to clean cold dry air, cold air + SO(2), and cold air + NO(2) produced weaker functional effects on the lower respiratory tract of guinea pigs than our previous consecutive 10-min exposures to these air conditions. After the first 10 min, there was a strong attenuation of the bronchoconstrictor responses, especially to cold air + NO(2) at 1 ppm. The small airway effects of prolonged mechanical ventilation were significantly modified by NO(2) at 1 ppm in both cold dry and warm humid breathing air. Finally, cold air + NO(2) at 1 ppm increased the proportion of macrophages in BALF white cells.

A new guinea-pig model for acute inhalation studies on combined effects of cold air and pollutants.

We have developed a new small animal model for acute inhalation studies on combined effects of cold air and gaseous urban air pollutants. The anaesthetised, tracheostomised and paralysed guinea-pig was placed inside a small, sealed whole-body-box, in which it was ventilated mechanically by using cyclic negative pressure (Pbox) for active expansion of the chest. During a 2-h normal ventilation with warm humid air (n=6), there was a need for increasing Pbox with time to maintain the fixed tidal volume (VT) of 11 ml/kg. No such need was seen in the experiments with 15-min periods of isocapnic hyperventilation at 80 and 120 breaths/min (n=13). During the 2-h normal ventilation and in experiments with hyperventilation, there was a gradual increase in heart rate and small gradual decreases in PaCO2 and pH with time. Cold air + SO2 2.5 ppm produced a significantly stronger bronchoconstriction (deltaVT=-30.3+/-7.2%, n=6, P < 0.05) than clean cold dry air (deltaVT=-10.6+/-1.3%, n=6) and cold air + NO2 2.5 ppm (deltaVT=-13.2+/-3.3%, n=6), although these three exposure conditions produced similar decreases in tracheal air and retrotracheal tissue temperatures. With the present guinea-pig model, the combined respiratory effects of cold air and gaseous urban air pollutants can be investigated in a highly controlled manner.